Prevascularization of a Gas-Foaming Macroporous Calcium Phosphate Cement Scaffold Via Coculture of Human Umbilical Vein Endothelial Cells and Osteoblasts

Abstract

The lack of a vasculature in tissue-engineered constructs is currently a major challenge in tissue regeneration. There has been no report of prevascularization of macroporous calcium phosphate cement (CPC) via coculture of endothelial cells and osteoblasts. The objectives of this study were to (1) investigate coculture of human umbilical vein endothelial cells (HUVEC) and human osteoblasts (HOB) on macroporous CPC for the first time; and (2) develop a new microvasculature-CPC construct with angiogenic and osteogenic potential. A gasfoaming method was used to create macropores in CPC. HUVEC and HOB were seeded with a ratio of HUVEC:HOB = 4:1, at 1.5 · 10 5 cells/scaffold. The constructs were cultured for up to 42 days. CPC with a porosity of 83% had a flexural strength (mean -SD; n = 6) of 2.6 -0.2 MPa, and an elastic modulus of 340 -30 MPa, approaching the reported values for cancellous bone. Reverse transcription-polymerase chain reaction showed that HUVEC + HOB coculture on CPC had much higher vascular endothelial growth factor (VEGF) and collagen I expressions than monoculture ( p < 0.05). Osteogenic markers alkaline phosphatase, osteocalcin (OC), and runt-related transcription factor 2 (Runx2) were also highly elevated. Immunostaining of PECAM1 (CD31) showed abundant microcapillary-like structures on CPC in coculture at 42 days, as HUVEC self-assembled into extensive branches and net-like structures. However, no microcapillary was found on CPC in monoculture. In immunohistochemical staining, the neo-vessels were strongly positive for PECAM1, the von Willebrand factor, and collagen I. Scanning electron microscopy revealed microcapillary-like structures mingling with mineral nodules on CPC. Cell-synthesized minerals increased by an order of magnitude from 4 to 42 days. In conclusion, gas-foaming macroporous CPC was fabricated and HUVEC + HOB coculture was performed for prevascularization, yielding microcapillary-like structures on CPC for the first time. The novel macroporous CPC-microvasculature construct is promising for a wide range of orthopedic applications with enhanced angiogenic and osteogenic capabilities.

Prevascularization of biofunctional calcium phosphate cement for dental and craniofacial repairs

“…Several studies showed that coculturing endothelial cells with osteoblasts-like cells resulted in the formation of microcapillary-like structures similar to those observed in vivo [17,18]. Although CPC is promising for bone repair due to its excellent biocompatibility and bone replacement capability, little has been reported on prevascularization of CPC, with only one report on coculturing endothelial cells and osteoblasts to prevascularize CPC [23]. The coculture could promote the self-assembled organization of the microcapillary-like structures.…”

“…Another study used starch-based scaffold to coculture osteoblasts and endothelial cells and obtained microcapillary-like structures [17]. However, a literature search revealed no report on prevascularization of CPC, except our recent study on coculture of endothelial cells and osteoblasts on CPC without biofunctionalization [23], in which cell attachment was not robust.…”

Overcoming physical constraints in bone engineering: ‘the importance of being vascularized’

“…Establishing tridimensional co-cultures embedded in a variety of scaffolds may improve our knowledge of bone remodeling and possibly enable future clinical application (Brennan et al, 2013;Deb et al, 2010;Dorst et al, 2014;Gao et al, 2014;Hofmann et al, 2008;Santos et al, 2009;Thein-Han & Xu, 2013). In particular, co-cultures within scaffolds under proper environmental stimuli have been proposed to explore the interaction of ECs with other cell types already seeded in the scaffold or attracted by the surrounding implanted (hosting) tissue.…”

“…In the last few years, research has been prompted to develop increasingly complex 3D culture systems in bioreactors, where cells are seeded on proper scaffolds and stimulated so as to mimic the physiological conditions more accurately. These bioreactors aim at enabling bone regeneration by incorporating different cells types, such as osteoblasts and endothelial cells (ECs), into bioinspired materials within a surveilled habitat (Brennan, Davaine, & Layrolle, 2013;Deb, Mandegaran, & Di Silvio, 2010;Dorst, Oberringer, Grässer, Pohlemann, & Metzger, 2014;Gao et al, 2014;Hofmann et al, 2008;Santos et al, 2009;Thein-Han & Xu, 2013). …”

Engineering 3D Models of Tumors and Bone to Understand Tumor-Induced Bone Disease and Improve Treatments

“…Traditional methods to fabricate porous TECs from natural or synthetic polymers include gas foaming [62, 63], particulate leaching [52, 64, 65], or freeze-drying [50, 66]. While these methods are effective in creating porous scaffolds, they lack the control necessary to create specific architectures.…”